Topologically Controlled Syntheses of Unimolecular Oligo[n]catenanes

Catenanes are a well-known class of mechanically interlocked molecules that possess chain-like architectures and have been investigated for decades as molecular machines and switches. However, the synthesis of higher-order catenanes with multiple, linearly interlocked molecular rings has been greatly impeded by the generation of unwanted oligomeric byproducts and figure-of-eight topologies that compete with productive ring closings. Here, we report two general strategies for the synthesis of oligo[n]catenanes that rely on a molecular “zip-tie” strategy, where the “zip-tie” is a central core macrocycle precursor bearing two phenanthroline (phen) ligands to make odd-numbered oligo[n]catenanes, or a preformed asymmetric iron(II) complex consisting of two macrocycle precursors bearing phen and terpyridine ligands to make even-numbered oligo[n]catenanes. In either case, preformed macrocycles or [2]catenanes are threaded onto the central “zip-tie” core using metal templation prior to ring-closing metathesis (RCM) reactions that generate several mechanical bonds in one pot. Using these synthetic strategies, a family of well-defined linear oligo[n]catenanes were synthesized, where n = 2, 3, 4, 5, or 6 interlocked molecular rings, and n = 6 represents the highest number of linearly interlocked rings reported to date for any isolated unimolecular oligo[n]catenane.


Section A. Materials / General Methods / Instrumentation
All reagents were purchased from commercial suppliers and used without further purification unless stated otherwise. Metal sources Fe(BF4)2•6 H2O, Cu(MeCN)4PF6, and CuI were purchased from Sigma-Aldrich. FeCl2 was purchased from Fisher Scientific. Modified literature procedures were employed in the synthesis of compounds F, G, 2, and Fe-(2)2. 1 All reactions were performed under N2 using common Schlenk techniques. Column chromatography was carried with silica gel (Sorbtech, 0.040 -0.063 mm) or neutral alumina (Sorbtech, Act. 1, 0.050 -0.2 mm) or basic alumina (Sorbtech, Act. 1, 0.050 -0.2 mm). All ring-closing reactions were done using Grubbs' 2 nd generation catalyst (Chem Scene). Recycling preparative gel permeation chromatography (prep-GPC) was performed on a Japan Analytical Industry LaboACE instrument with one GEL-2HR column and one JAIGEL-2.5HR column in sequence, running with dimethylformamide (DMF) at 8 mL‧min -1 as the mobile phase. All nuclear magnetic resonance (NMR) spectra were recorded on Varian Inova-500 spectrometer at 25 °C, with working frequencies of 500 ( 1 H) and 125 ( 13 C) MHz. Chemical shifts are reported in ppm relative to the signals corresponding to the residual non-deuterated solvents: CDCl3: δH = 7.26 ppm and δC = 77.16 ppm. Ultraviolet-Visible (UV-Vis) absorbance spectra were recorded on an Agilent Cary 5000 spectrophotometer with a quartz cuvette (1.0 cm pathlength). Analytical GPC analyses were performed on an Agilent 1260 Infinity setup with two Shodex GPC KD-806M columns in sequence in DMF mobile phase (0. 025 M LiBr) running at 60 °C at 1.0 mL‧min -1 . The differential refractive index (dRI) of each compound was monitored using a Wyatt Optilab T-rEX detector. Analytical HPLC analyses were performed on an Avant 2000 HPLC with a Shodex Asahipak ODP-50-2D reverse-phase column with a gradient mobile phase of H2O with 0.1% TFA and MeCN with 0.1% TFA running at 40 °C at 0.2 mL‧min -1 , which was in series with an Advion Expression-L Compact Mass Spectrometer; UV-vis absorbance was recorded at 254 nm. Low-resolution mass spectrometry electrospray ionization (LRMS-ESI) was recorded on an Advion Expression-L Compact Mass Spectrometer. High-S4 resolution mass spectrometry electrospray ionization (HRMS-ESI) was recorded on a Waters Synapt G2 HDMS or a Bruker maXis 4G UHR-TOF mass spectrometer. Tandem high-resolution mass spectrometry electrospray ionization (THRMS-ESI) was recorded on a Bruker maXis 4G Q-TOF mass spectrometer. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was recorded on a Bruker Solaris 12T FT-MS; samples were prepared using 2,5-dihydroxybenzoic or α-cyano-4-hydroxycinnamic acid matrices.

1) Synthesis of Open and Closed Macrocyclic Ligands a) Mono-Allyl Diethylene Glycol Mesyl (A)
Scheme S1. Synthesis of compound A.
Step 1: A solution of diethylene glycol (25.00 g, 22.3 mL, 235.6 mmol, 1.0 eq.) was prepared in 300 mL tetrahydrofuran (THF) in a 1 L round-bottom (RB) flask with a stir bar. While stirring, a solution of NaOH (6.59 g, 164.9 mmol, 0.7 eq.) in 25 mL DI H2O was added. The solution was cooled using an ice bath for 1 h before adding a solution of allyl bromide (34.2 g, 24.4 mL, 282.7 mmol, 1.2 eq.) in 200 mL THF via slow addition funnel over the course of several hours. The reaction was allowed to warm up to room temperature while stirring open to air for 5 d. The solvent was then removed via rotary evaporator. The crude oil was taken up in 1L brine and 500 mL dichloromethane (CH2Cl2). The aqueous layer was further extracted with 4 x 500 mL CH2Cl2. The S5 organics were dried over Na2SO4 and filtered. The solvent was removed via rotary evaporator to afford the crude asymmetric product as an orange oil, which was taken forward to mesylation without further purification.
Step 2: The crude orange oil was redissolved in 500 mL CH2Cl2 in a 1 L RB flask with a stir bar.

Scheme S2. Synthesis of compound B.
Step 1: A suspension of phenanthroline (phen) diol 1 and Cs2CO3 was prepared in 200 mL DMF in a 500 mL RB flask. The suspension was heated to 55 °C for 1 h while stirring under N2 before adding a solution of A (0.990 g, 4.41 mmol, 1 eq.) in 25 mL DMF via syringe pump at 3 mL‧h -1 .
After heating for a total of 6 d, the solvent was removed via rotary evaporator. The crude was taken up in 300 mL 10% MeOH / CH2Cl2. The suspension was filtered via vacuum filtration over a 150 mL fritted funnel with medium porosity. The remaining solid was washed with 200 mL 10% MeOH / CH2Cl2. The filtrate was concentrated via rotary evaporator and was again taken up in 200 mL 10% MeOH / CH2Cl2. The resultant solid was again collected via vacuum filtration and was kept to later repurify the phen diol starting material. The filtrate was concentrated into a 100 mL high pressure vessel using a rotary evaporator. The sticky orange crude solid was dried on high vacuum within the high-pressure vessel for 1 h before proceeding to the next step.
A solution of triethylene glycol (25.00 g, 150.2 mmol, 1.0 eq.) was prepared in 300 mL THF in a 1 L round-bottom (RB) flask with a stir bar. While stirring, a solution of NaOH (4.66 g, 116.5 mmol, 0.7 eq.) in 25 mL DI H2O was added. The solution was cooled using an ice bath before adding a solution of allyl bromide (24.2 g, 17.3 mL, 199.8 mmol, 1.2 eq.) in 200 mL THF via slow addition funnel over the course of several hours. The reaction was allowed to warm up to room temperature while stirring open to air for 5 d. The solvent was then removed via rotary evaporator and the crude yellow sludge was taken up in 500 mL CH2Cl2. The organic layer was washed with 2 x 200 mL DI H2O and 2 x 200 mL brine. The organics were dried over Na2SO4 and filtered. The solvent was removed via rotary evaporator to afford the product as a light yellow oil (10.5 g, 37%). 1 H NMR (500 MHz, CDCl3): δH 1H),2H),4.03 (d,J = 5.7 Hz,2H),2H),6H),4H). 13 C NMR (125 MHz, CDCl3): δC 134.7, 117.1, 72.5, 72.3, 70.7, 70.6, 70.4, 69.4, 61.7.

Scheme S5. Synthesis of compound D.
A solution of C (10.47 g, 55.1 mmol, 1.0 eq.) and Et3N (29.2 g,40.0 mL,288.6 mmol,5.2 eq.) was prepared in 500 mL CH2Cl2 in a 2 L RB flask fitted with a slow addition funnel and containing a stir bar. The solution was cooled using an ice bath before adding a solution of mesyl chloride (MsCl) (19.2 g, 13.0 mL, 167.9 mmol, 3.0 eq.) in 300 mL CH2Cl2 via slow addition funnel over the course of 2 h. The reaction was allowed to warm up to room temperature while stirring overnight.
After 16 h, the crude was washed with 5 x 200 mL 1M HCl. The organics were dried over Na2SO4 and filtered. The solvent was removed via rotary evaporator to afford the crude as an orange oil. (m, 8H), 3.00 (s, 3H). 13 C NMR (125 MHz, CDCl3): δC 134. 6, 116.8, 71.9, 70.39, 70.37, 70.32, 69.3, 69.2, 68.8, 37.5 A suspension of terpyridine (terpy) diol 1 (1.50 g, 5.65 mmol, 1.0 eq.) and Cs2CO3 (9.2 g, 28.3 mmol, 5 eq.) was prepared in 200 mL DMF in a 1L RB flask with a stir bar. While stirring under N2, the suspension was heated to 75 °C before adding a solution of D (4.6 g, 16.95 mmol, 3 eq.) in 300 mL DMF via slow addition funnel over the course of 1 h. The reaction temperature was kept at 75 °C while stirring under N2 for 2 d. The reaction mixture was allowed to cool to room temperature and was filtered via gravity filtration to remove insoluble inorganic salts. The filtrate was concentrated via rotary evaporator. The crude was resuspended in 300 mL CH2Cl2 and was washed with 3 x 100 mL brine. The organics were dried over Na2SO4 and filtered. The solvent was removed via rotary evaporator to afford the crude as a yellow oil. Basic alumina column chromatography (50% hexanes / CH2Cl2 to 1% MeOH / CH2Cl2) of the crude material afforded the product as a bright yellow oil (2.9 g, 85%). 1 H NMR (500 MHz, CDCl3): δH 8.54 (d, J = 8.8 Hz,  5, 155.1, 149.5, 137.8, 137.2, 134.9, 122.0, 121.8, 119.7, 117.20, 72.4, 71.1, 70.8, 69.8, 69.6, 68. We have previously reported 1 the synthesis of compound F. Herein, we report an improved synthetic method; terpy diol and mono tosylated bis(triethylene glycol) hydroquinone were prepared according to our previous work. 1 A suspension of terpy diol (1.51 g, 5.68 mmol, 3.0 eq.) and Cs2CO3 (9.3 g, 28.4 mmol, 15.0 eq.) was prepared in 250 mL DMF in a 500 mL RB flask. The suspension was heated to 100 °C while stirring under N2 for 1h. The reaction mixture was then cooled to 75 °C before adding a solution of mono tosylated bis(triethylene glycol) hydroquinone (1.00 g, 1.91 mmol, 1.0 eq.) in 150 mL DMF via slow addition over several hours. The reaction temperature was kept at 75 °C while stirring under N2 for a total of 4 d. The reaction mixture was then allowed to cool to room temperature and the suspension was filtered via gravity filtration to remove solid inorganic salts. The solvent was then removed via rotary evaporator to afford the crude as a black oily mixture. To the crude was added 300 mL 1M CH3COOH. The suspension was sonicated and then stirred at room temperature for 1 day before dilution with 300 mL CH2Cl2.
The aqueous layer was then extracted with 3 x 100 mL CH2Cl2. The organics were dried over Na2SO4 and were filtered. The solvent was then removed via rotary evaporator to afford the crude product as a brown oil. The crude oil was redissolved in 18 mL GPC grade DMF and was purified via recycling prep-GPC with DMF over five injections to afford the asymmetric product as a brown oil (0.57 g, 49%). 1 H NMR is consistent with our previous work.

Scheme S8. Synthesis of compound G.
We have previously reported 1 the synthesis of compound G. Herein, we report an improved synthetic protocol; phen diol and mesylated triethylene glycol hydroquinone olefin were prepared according to our previous work. 1 A suspension of phen diol (3.30 g, 9.05 mmol, 2.5 eq.) and Cs2CO3 (1.73 g, 5.30 mmol, 1.5 eq.) was prepared in 250 mL DMF in a 500 mL RB flask and was heated to 60 °C while stirring under N2. A solution of mono mesylated triethylene glycol hydroquinone olefin (1.33 g, 3.56 mmol, 1.0 eq.) in 50 mL anhydrous DMF was added via syringe pump at 3 mL‧h -1 . After heating for a total of 5 d, the reaction solvent was removed via rotary evaporator. To the crude was added 400 mL 10% MeOH / CH2Cl2. The suspension was stirred at room temperature overnight. The suspension was then filtered via vacuum filtration over a 150 mL fritted funnel with medium porosity. The filtrate was concentrated via rotary evaporator. The crude sludge was taken up in 300 mL CH2Cl2, which caused unreacted phen diol starting material to precipitate as an orange solid. The starting material was collected via vacuum filtration. The filtrate was concentrated and was purified via silica column chromatography with a very slow gradient (CH2Cl2 to 2 % MeOH / CH2Cl2). The product was recovered as a sticky orange solid (0.87 g, 38%). 1 H NMR is consistent with our previous work.

Scheme S9. Synthesis of compound 2.
We have previously reported 1 the synthesis of 2. Herein, we report an improved and scaled synthetic protocol; mesylated terpyr olefin was prepared according to our previous work. 1 Compound G (0.55 g, 0.86 mmol, 1.2 eq.) and mesylated terpy olefin (0.54 g, 0.715 mmol, 1.0 eq.) was transferred to a 100 mL high-pressure flask (Kemtech) using a minimal amount of CH2Cl2. The solvent was removed via rotary evaporator and the resulting foam was dried on high vacuum for 2 h. Solid Cs2CO3 (1.16 g, 3.56 mmol, 5.0 eq.) was added to the vessel, followed by the addition of 40 mL anhydrous MeCN and a stir bar. The vessel was sealed with a Teflon screw cap and was heated to 100 °C for 1 d. After 1 d, the reaction was allowed to cool to room temperature. The crude suspension was diluted with 400 mL CH2Cl2 and was washed with 3 x 100 mL brine. The organics were dried over Na2SO4 and were filtered. The solvent was removed via rotary evaporator to afford crude product as an orange film. Basic alumina column chromatography (CH2Cl2 to 3% MeOH / CH2Cl2) of the crude material afforded the product as a sticky orange solid (0.66 g, 71%). 1 H NMR is consistent with our previous work.

Scheme S10. Synthesis of compound H.
Step 1: A suspension of terpy diol 1 (0.50 g, 1.87 mmol, 1.0 eq.) and Cs2CO3 (3.71 g, 11.38 mmol, 6.0 eq.) was prepared in 400 mL DMF in a 1 L RB flask fitted with a slow addition funnel. A solution of mono tosylated bis(triethylene glycol) hydroquinone (2.98 g, 5.65 mmol, 3.0 eq.) in 200 mL DMF was added via slow addition over 2 h. The reaction was heated to 75 °C while stirring under N2. After 16 h, the solvent was removed by rotary evaporator and the crude product was taken up in 300 mL CH2Cl2. The organics were washed with 3 x 100 mL brine, dried over Na2SO4, and filtered. The filtrate was concentrated via rotary evaporator and was transferred to a 50 mL centrifuge tube. Et2O was added to precipitate the product out of solution as a white solid and the product was collected via centrifugation. The supernatant was decanted off and the white solid pellet was redissolved in a minimal amount of CH2Cl2. The precipitation and centrifugation processes were repeated a second time. The pellet was then dried on high vacuum and was taken forward to mesylation without further purification.

S15
Step 2: Stoichiometry assumes full conversion for previous step. A solution of crude terpy hydroquinone triethylene glycol (1.87 mmol, 1.0 eq.) and Et3N (2.85 g, 3.9 mL, 28.2 mmol, 15 eq.) was prepared in 150 mL CH2Cl2 in a 500 mL RB flask fitted with a stir bar. The solution was cooled using an ice bath under N2 before adding a solution of mesyl chloride (MsCl) (2.15 g, 1.45 mL, 18.76 mmol, 10 eq.) in 100 mL CH2Cl2 via slow addition funnel. The reaction was allowed to warm up to room temperature while stirring under N2 for 2 d. The solution was then diluted with 300 mL CH2Cl2 and was washed with 3 x 100 mL 1 M CH3COOH and 2 x 100 mL aqueous saturated NaHCO3. The organics were dried over Na2SO4 and were filtered. The solvent was removed via rotary evaporator. The crude was redissolved in 25 mL GPC grade DMF and was filtered. It was then purified via recycling prep-GPC with DMF over five injections to afford the product as a white solid (0.97 g, 45% over two steps). 1  Scheme S11. Synthesis of compound 3.
We have previously reported 1 the synthesis of Fe-(2)2. Herein, we report an improved and scaled synthetic protocol. A solution of 2 (0.500 g, 0.385 mmol, 1 eq.) was prepared in 150 mL THF in a 250 mL RB flask. The flask was purged with N2 and a solution of Fe(BF4)2 • 6 H2O (0.169 g, 0.499 mmol, 1.3 eq.) in 25 mL DI H2O was added via syringe. The dark red solution was heated at 60 °C for 1 h while stirring under N2. The solution was then allowed to cool to room temperature before diluting with 300 mL CH2Cl2. The dark red solution was washed with 3 x 100 mL brine. The aqueous layer was back-extracted with 2 x 100 mL CH2Cl2. The combined organics were dried over Na2SO4 and were filtered. The solvent was removed via rotary evaporator to afford the product as a dark red sticky solid (0.542 g, 99%). 1 H NMR is consistent with our previous work. S19 b) [2]Catenane-TPM ( [2]C) and Figure-Eight TPM (I) Scheme S13. Synthesis of compounds [2]C and I.
Step 1: A solution of Fe-(2)2 (0.209 g, 0.074 mmol, 1 eq.) and Grubbs' 2 nd generation catalyst (0.012 g, 0.014 mmol, 0.2 eq.) was prepared in 200 mL CH2Cl2 in a 500 mL RB fitted with a Vigreux column. The dark red solution was then heated to 35 °C while stirring under N2. The reaction progress was monitored by LR-ESI. After 1 d, the reaction was complete and was quenched with 1 mL ethyl vinyl ether (EVE) and 5 mL MeCN. The solvent was removed to afford the crude product as a dark red film. The crude material was purified via column chromatography with basic alumina (CHCl3 to 5% MeOH / CHCl3). All fractions, including mixed fractions, containing the desired product were collected to be demetalated prior to purification via recycling preparative GPC.
Step 2: The mixed fractions were redissolved in 25 mL DMF in a 50 mL RB flask with a stir bar.
A solution of 3 (0.257 g, 0.197 mmol, 1 eq.) was prepared in 130 mL THF in a 250 mL RB flask.

S24
Step 1: A solution of diallyl terpy E (0.271 g, 0.444 mmol, 2.0 eq.) and 3 (0.2817 g, 0.216 mmol, 1.0 eq.) was prepared in 300 mL THF in a 500 mL RB flask. While stirring under N2, a solution of Fe(BF4)2 • 6 H2O (0.220 g, 0.65 mmol, 3.0 eq.) in 50 mL DI H2O was added via syringe. The dark red solution was then heated to 60 °C for 1 h while stirring under N2. The solution was then allowed to cool to room temperature while stirring for 2 d. The solvent was removed via rotary evaporator and crude material was taken up in 300 mL CH2Cl2. The dark red solution was washed with 3 x 100 mL DI H2O. The organics were dried over Na2SO4 and were filtered. The solvent was removed via rotary evaporator and the crude product was isolated as a dark red film, which was taken forward to the next step without any further purification.
Step 2: The crude red film was redissolved in 500 mL CH2Cl2 in a 1 L RB flask. A solution of Grubbs' 2 nd generation catalyst (0.018 g, 0.0216 mmol, 0.1 eq.) in 5 mL CH2Cl2 was added. The reaction was fitted with a Vigreux column and was heated to 35°C while stirring under N2. The reaction progress was monitored by LR-ESI. After 8 h, an additional 0.05 eq. Grubbs' 2 nd generation catalyst was added, and the reaction temperature was maintained at 35°C while stirring under N2. After 1 d of total heating, the reaction was complete and was quenched with 5 mL EVE and 5 mL MeCN. The solvent was removed via rotary evaporator and the crude was transferred to a 100 mL RB flask.
Step 1: A solution of [2]C-E (0.0701 g, 0.0371 mmol, 1 eq.) was prepared in 5 mL C2H4Cl2 in a 10 mL high-pressure vessel with a stir bar. The vessel was then charged with B2(OH)4 (0.341 g, 3.80 mmol, 100 eq.), N-methylmorpholine (0.75 g, 0.82 mL, 7.43 mmol, 200 eq.) and 10% Pd on carbon (0.035 g, 0.5 eq. wt/wt ). 3 It was quickly sealed and was heated to 45°C while stirring for 1 d. The reaction was then allowed to cool to room temperature. The catalyst was removed via gravity filtration and the black solid was washed with 100 mL 10% MeOH / CH2Cl2. The filtrate was washed with 3 x 50 mL brine. The organics were dried over Na2SO4 and were filtered. The solvent was removed rotary evaporator and the crude yellow film was taken forward to the rearomatization step without further purification.

Scheme S17. Synthesis of compound Fe-[2]C-E.
A solution of Fe- [2]C-E (0.183 g, 0.0968 mmol, 1 eq.) was prepared in 100 mL 25% MeOH / THF in a 100 mL RB flask. The flask was purged with N2 and a solution of Fe(BF4)2 • 6 H2O (0.0507 g, 0.1501 mmol, 1.5 eq.) in 10 mL DI H2O was added via syringe. The dark red solution was heated to 60°C for 1 h while stirring under N2. The solution was then allowed to cool to room temperature and the solvent was removed via rotary evaporator. The crude product was taken up in 300 mL CH2Cl2 and was washed with 3 x 100 mL DI H2O. The aqueous layer was back-extracted with 3 x 100 mL CH2Cl2. The combined organics were dried over Na2SO4 and were filtered. The solvent was removed via rotary evaporator to afford a dark red film. The crude product was redissolved S29 in a minimal amount of CH2Cl2 and was transferred to a 50 mL centrifuge tube. The product was precipitated out of solution by the addition of hexanes. The red suspension was centrifuged, and the supernatant was discarded. The dark red pellet was redissolved in CH2Cl2 and was again precipitated out of solution by the addition of hexanes. The red suspension was centrifuged, and the supernatant was discarded. The dark red pellet was redissolved in CH2Cl2 and was transferred to a vial. The solvent was removed via rotary evaporator to afford the product as a dark red solid  3, 159.59, 159.56, 157.6, 157.5, 153.3, 153.2, 149.96, 149.94, 146.1, 141.8, 140.9, 138.9, 138.4, 137.1, 132.3, 129.0, 127.7, 125.9, 125.3, 124.9, 124.1, 122.3, 121.9, 121.2, 119.4, 115.86, 115.80, 115
Step 1: A solution of Fe-(3)2 (1.08 g, 0.38, mmol, 4.0 eq.) was prepared in 100 mL MeCN in a 500 mL RB flask with a stir bar. While stirring at 25 °C under N2, a solution of CuI (0.135 g, 0.71 mmol, 7.5 eq.) in 50 mL MeCN was added. After stirring for 1 h, a solution of 1 (0.124 g, 0.096 mmol, 1.0 eq.) in 25 mL CH2Cl2 was added via syringe. The reaction was continued to stir at 25 °C for 16 h. The solvent was then removed via rotary evaporator and the crude dark red film was dried on high vacuum for several hours before ring-closing.
A solution of Grubbs' 2 nd generation catalyst (0.008 g, 0.0095 mmol, 0.1 eq.) was added and the S31 RB flask was fitted with a Vigreux column. The reaction was heated to 35 °C while stirring under N2 for 3 d. An extra 0.1 eq. of Grubbs' 2 nd generation catalyst was added, and the reaction was continued to heat for an additional 1 d. The reaction was then quenched with EVE and the solvent was removed via rotary evaporator.
The dark red solution was washed with 3 x 25 mL DI H2O. The organics were dried over Na2SO4 and were filtered. The solvent was removed via rotary evaporator to afford the pre- [4]catenate complex as a foamy red solid, which was taken forward to the next step without further purification.
Step 2: The crude foamy red solid was redissolved in 60 mL anhydrous CH2Cl2 in a 250 mL RB flask with a stir bar. A solution of Grubbs' 2 nd generation catalyst (0.004 g, 0.0045 mmol, 0.2 eq.) in 1 mL CH2Cl2 was added. The flask was fitted with a Vigreux column, and the dark red solution was heated to 35°C while stirring under N2. After 1 d, an additional 0.2 eq. Grubbs' 2 nd generation catalyst was added. The reaction was heated at 35°C while stirring under N2 for an additional 1 d.
After 2 d of total heating, the reaction was quenched with 1 mL EVE and 5 mL MeCN. The solvent was removed via rotary evaporator to afford the crude [4]catenate mixture as a dark red film.
Step 3: a) The Fe 2+ ion was first removed from the crude mixture with the addition of a weak inorganic base and moderate heating. The crude red film was redissolved in 50 mL DMF in a 250 mL RB flask. Solid Cs2CO3 (1.0 g, 3.07 mmol, 135 eq.) was added and the suspension was heated to 75°C for 1 d while stirring open to air. b) The Cu + then removed by addition of an excess of strongly competing ligand, namely KCN. The solvent was then removed via rotary evaporator and 50 mL MeCN was added to the crude suspension. A solution of KCN (0.2 g, 3.07 mmol, 135 eq.) in 10 mL H2O was added via syringe. The suspension was stirred at room temperature for 30 min before dilution with 300 mL CH2Cl2. The organics were washed with 3 x 50 mL brine. The aqueous layer was back-extracted with 2 x 100 mL CH2Cl2. The combined organics were dried over Na2SO4 and were filtered. The filtrate was concentrated via rotary evaporator to afford the crude product as a yellow/orange film. The crude was redissolved in 5 mL GPC grade DMF and the solution was centrifuged to remove any remaining insoluble salts. The supernatant was filtered via syringe filter and was purified via recycling prep-GPC with DMF to afford only mixed [4]catenane product.
Step 2: The crude pre- [5]catenate complex was redissolved in 25 mL anhydrous CH2Cl2 in a 50 mL RB flask. A solution of Grubbs' 2 nd generation catalyst (0.002 g, 0.0023 mmol, 0.2 eq.) was added and the RB flask was fitted with a Vigreux column. The reaction was heated to 35 °C while stirring under N2. After 16 h, an additional 0.1 eq. Grubbs' 2 nd generation catalyst was added and the reaction was continued to heat at 35 °C for 10. The reaction was quenched with EVE and the solvent was removed via rotary evaporator.
Step 1: A solution of Cu(MeCN)4PF6 (0.0105 g, 0.028 mmol, 4.0 eq.) in 3 mL N2-purged anhydrous MeCN was added via syringe to a solution of Fe- [2]C-E (0.0601 g, 0.0284 mmol, 4.0 eq.) in 10 mL N2-purged anhydrous MeCN in an oven dried 50 mL RB flask. The dark red solution was stirred at room temperature under N2. After 30 min, a solution of Fe-(2)2 (0.022 g, 0.0077 mmol, S38 1.0 eq.) in 7 mL N2-purged anhydrous MeCN was added via syringe. The dark red solution continued to stir at room temperature under N2 for 2.5 d. The solvent was then removed via rotary evaporator to afford the crude product as a foamy red solid, which was taken up in 100 mL CH2Cl2.
The organic layer was washed with 3 x 50 mL DI H2O and was dried over Na2SO4. The dark red solution was filtered, and the solvent was removed via rotary evaporator to afford the pre- [6]catenate complex as a dark red film, which was used in the next step without further purification.
Step 2: The crude foamy red solid was redissolved in 25 mL anhydrous CH2Cl2 in a 100 mL RB flask with a stir bar. A solution of Grubbs' 2 nd generation catalyst (0.0013 g, 0.0015 mmol, 0.2 eq.) in 1 mL CH2Cl2 was added. The flask was fitted with a Vigreux column, and the dark red solution was heated to 35°C while stirring under N2. After 18 h, an aliquot was quenched with EVE and the reaction was deemed complete by 1 H NMR. The remaining solution was quenched with 1 mL EVE and 5 mL MeCN. The solvent was removed via rotary evaporator to afford the crude [6]catenate mixture as a dark red film.
Step 3: a) The Fe 2+ ion was first removed from the crude mixture with the addition of a weak inorganic base and moderate heating. The crude red film was redissolved in 25 mL DMF in a 100 mL RB flask. Solid K2CO3 (1.0 g, 7.23 mmol, 1000 eq.) was added and the suspension heated to               The degrees of freedom afforded by the mechanical bonds lead to complex shielding and deshielding of protons, which cause apparent subpopulations of peaks and lead to difficulty in the integration of specific protons. However, these subpopulations are not due to lack of purity. The purity of [4]C was confirmed by recycling preparative GPC (Fig. S58), analytical GPC (Fig. S51), and analytical HPLC (Fig. S61), and the product identities confirmed by MALDI-TOF (Fig. S72).      As the temperature is lowered, many of the sharp peaks observed at 25 °C begin to broaden. This is likely due to the conformational motions becoming more restricted at lower temperatures.                  . 1 equivalent of the Cu + source was added per ligand in each catenane (i.e., 4 eq. Cu + for [2]C, 8 eq. Cu + for [4]C, and 10 eq. Cu + for [6]C) was added directly to the cuvette. The sample was inverted 3 times and the metalated spectrum was recorded.

2) 2D 1 H-1 H NMR Spectra
This experiment was done in order to demonstrate that the isolated catenanes could be remetalated with Cu + . However, it should be noted that this metalation will not exclusively form Cu + -bis(phen) complexes because heteroleptic Cu + -phen-terpy complexes are also favorable 4 because both species are 18 electron complexes. The selectively of orthogonal metalation during the synthesis of these catenanes was achieved by first blocking the terpy ligands with Fe 2+ , followed by metalation with Cu + .      Despite being topological products with the same chemical formula and molecular weight, the figure-eight TPM I and the [2]catenane TPM [2]C adopt different conformations in solution. Such differences in apparent size make it possible to separate these topological products by size exclusion chromatography. Leigh and coworkers have recently utilized recycling preparatory GPC for the separation of twisted [2]catenane and molecular granny knot topological products. 5 Figure S57. Recycling preparatory GPC trace of [3]C using one JAIGEL-2HR column and one JAIGEL-2.5HR column in sequence with a DMF mobile phase at 8 mL‧min -1 . This trace was from the second GPC purification. Pure product was isolated in the green fraction at 108 min. Figure S58. Recycling preparatory GPC trace of [4]C using one JAIGEL-2HR column and one JAIGEL-2.5HR column in sequence with a DMF mobile phase at 8 mL‧min -1 . This trace was from the second GPC purification. Pure product was isolated in the light blue fraction at 235 min. Figure S59. Recycling preparatory GPC trace of [5]C using one JAIGEL-2HR column and one JAIGEL-2.5HR column in sequence with a DMF mobile phase at 8 mL‧min -1 . Pure product was isolated in the light blue fraction at 75 min. Figure S60. Recycling preparatory GPC trace of [6]C using one JAIGEL-2HR column and one JAIGEL-2.5HR column in sequence with a DMF mobile phase at 8 mL‧min -1 . Pure product was isolated in the green fraction at 125 min. Mixed fractions were also re-purified for overall yield.  Fig. 4c, Fig. S51), and 1 H NMR (Figs. S9-S12, and S15-S26). These shoulders could be caused S84 by these compounds streaking on the column due to limited solubility in H2O and hydrophilic organic solvents (MeOH, MeCN) that are commonly used in the mobile phase on reverse-phase columns. The low solubility explanation also addresses why these seemingly polar molecules do not elute off the reverse-phase column until a gradient of nearly pure organic solvent is reached.

S82
Attempts at running the higher order catenanes on normal-phase HPLC columns also proved unsuccessful presumably due to their inherent polarity. Unlike the THRMS-ESI of [2]C, the fragmentation of I results in a complex mass spectrum. Since there are no susceptible bonds in the large macrocycle, the fragmentation appears to be random and unpredictable, and therefore gives a wide range of fragments. The mixed macrocycle [2]catenane and macrocycle fragments could only have originated from the linear [4]catenane [4]C, as opposed to a hypothetical linear [3]catenane resulting from a figure-of-eight precursor.   Zoomed-in THRMS-ESI mixed macrocycle [2]catenane fragment. (c) Zoom-in THRMS-ESI of macrocycle fragment. The subpopulation of peaks corresponds to unfragmented 3 flying as [M +2H] 2+ , which is distinguishable from the macrocycle by its charge state.